3-Isobutyl-1-methylxanthine (IBMX) and certain related xanthines inhibited the generation of inositol phosphates, the increase in levels of free cytosolic Ca2+ ([Ca2+]i) and the secretion of 5-hydroxytryptamine in a rat tumor mast cell line (RBL-2H3 cells) in response to dinitrophenylated BSA (DNP-BSA). This inhibition did not correlate with the potency of these compounds as antagonists of adenosine receptors or as phosphodiesterase inhibitors. Instead, the inhibition appeared attributable to competitive antagonism of DNP-BSA binding to the DNP-specific IgE. Thus, the xanthines inhibited binding of [125I]DNP-BSA to cell bound DNP-specific IgE with the same rank order of potency as their inhibition of responses to antigen. The extent of inhibition by xanthines was inversely related to the proportion of receptors that were occupied by DNP-specific IgE. IBMX did not inhibit the responses to any other stimulants which included aggregated ovalbumin or concanavalin A in cells that were primed with ovalbumin-specific IgE or oligomers of IgE and antibody to IgE receptors in cells that were left unprimed. The effects of xanthine on DNP-BSA responses were markedly dependent on the chemical structure. IBMX was much more potent than theophylline. Because DNP-specific IgE is widely used in studies of antigen-induced stimulation of mast cells some reinterpretation of earlier studies in which xanthines have been utilized appears necessary.